WIST iGEM
WIST iGEM
Result
INOCULATION
Dickeya fangzhongdai
Bacillus subtilis 168
If the experiment is positive, meaning that Bacillus subtilis has natural function for killing Dickeya fangzhongdai, their will be an apparent gap between the cross section between Bacillus subtilis and Dickeya fangzhongdai.
The image shows the initial stage of the plate where we cultivate Dickeya fangzhongdai Ph21 and Bacillus subtilis 168 on LB Agar plates for a duration of 12 hours.
The outcome indicates that Bacillus subtilis, on its own, lacks the ability to eliminate Dickeya fangzhongdai.
The experiment takes another step to to assess the performance and efficacy of Bacillus subtilis 168 against Dickeya fangzhongdai Ph21 without any prior alterations. In contrast to the previous experiment, this one is conducted directly on orchid leaves.
The experiment takes another step to to assess the performance and efficacy of Bacillus subtilis 168 against Dickeya fangzhongdai Ph21 without any prior alterations. In contrast to the previous experiment, this one is conducted directly on orchid leaves.
In order to assess the survival of Bacillus subtilis 168, two different application methods were employed: immersion of leaf samples and surface spraying of the bacterial suspension onto the leaves. After a 24-hour incubation period, the study aimed to determine the persistence of Bacillus subtilis 168 under each application method.
On Day 2, bacterial enumeration was done by applying Bacillus subtilis 168 (B+) to three plates and distilled water (B-) to one plate. Leaves were treated accordingly with B+ or B-, and bacterial samples from leaf squares were evenly spread on three LB Agar plates per treatment. On Day 3, bacterial colony counting was carried out.
The results are highly successful, as they clearly illustrate that Bacillus subtilis possesses the capacity to establish endosymbiosis with orchid leaves, rather than perishing.
PLASMID CONSTRUCTION
This figure shows that the vector we extract has the correct backbone size of about 7900bp
The first well is the vector that has not been digested.
The second well is the vector that has been digested by one-cutting side restriction enzyme.
The third well is the vector that has been digested by two-cutting side restriction enzymes.
The fourth well is the result of primer checking the ampicillin by using PCR.
Backbone of the plasmid Firstly, we construct the plasmid with golden gate technique, then we transform the plasmid into Bacillus subtilis W168 to express the gene and stored in E. coli DH5α.
We inserted our desired gene fragments into our plasmid through the restriction sites XhoI and Aflii.
Insert aiiA + Kill Switch Next, we insert the aiiA gene and MazE and MazF toxin (Kill Switch). After the Kill Switch is well-functioned, we use different concentrations of lactone to check the less amount that is needed for Kill Switch to function.
Insert LuxR + sspB and Degron System Lastly, we insert the LuxR with sspB and Degron system ( include gRNAs, dCas9, CI repressor and ssrA DAS+4 tag ) into the pWS4 plasmid, and we named this plasmid as pWS4-1. It has been completely constructed and is looking forward to verification.